Carboxamido nitrobenzene disulfide compound

ABSTRACT

Test compositions, indicators, and test device are provided which are capable of generating different hues at different analyte concentrations. The compositions are capable of generating a yellow hue in situ. Visual tests for clinically important analytes, such as glucose, are determined by use of two independent catalytic systems which are reactive with reduced nicotinamide adenine dinucleotide to produce a range of hues; the particular hue produced depending on the concentration of the analyte. The invention provides a test device for the determination of analyte, e.g. glucose, in body fluid which exhibits a RAINBOW of hues, the particular final hue produced depending on the analyte concentration.

This is a related application of application Ser. No. 79,692, filed onJuly 30, 1987, now U.S. Pat. No. 4,975,367, which was acontinuation-in-part of application Ser. No. 848,706, filed on Apr. 4,1986, now abandoned.

FIELD OF THE INVENTION

The invention relates to test compositions and test devices capable ofgenerating different hues at different analyte concentrations Visualtests for clinical analytes are the focus of the invention.

In particular, the invention provides a self-indicating test device forthe determination of analytes, e.g. glucose, in body fluid whichexhibits the colors of the RAINBOW.

UTILITY

Colorimetric tests are conveniently used as visual tests with whichrelatively untrained personnel can routinely obtain results by simplecomparison to an appropriate color chart. Visual tests are low cost andconvenient since no instrumentation is required Presently, visual testsare used for routine screening of urine samples for a number ofdiagnostically important analytes, used by diabetics for home testing ofurine or blood glucose and used in other fields, for example watertesting for iron content.

However, currently available tests relate the intensity of a particularcolor to the concentration of analyte. For example, a test device maychange from colorless to light blue to darker shades of blue withincreasing concentration of glucose. Greater visual discrimination, andtherefore greater accuracy, is possible, when a range of colors isprovided rather than different shades of a single color. Therefore atest composition which exhibits different colors at different analyteconcentrations would be easier to use and would provide more accuratevisual results.

In order to allow visual differentiation of higher concentrations ofglucose, many currently available products resort to the use of tworeagent pads, one of which provides better color differentiation at thehigher concentration range. Otherwise such products would exhibit onlyvery slightly differing shades of dark blue (or dark green) above 150mg/dL glucose In contrast, a test device of this invention can producedramatic color changes, blue to sea green to yellow to orange to red,over a range of 0 to 800 mg/dL of glucose.

This invention provides compositions which can be used to produce arange of colors, especially for clinically important analytes. Inparticular, a test device, capable of generating the hues of a fullspectrum RAINBOW, for the determination of glucose in a body fluid, suchas whole blood, is shown.

INFORMATION DISCLOSURE

U.S. Pat. No. 4,490,465 discloses a test system for the determination ofglucose having an extended range of measurement. The system involves atleast one pyridine linked dehydrogenase and one nonpyridine linkeddehydrogenase One example shows a determination of glucose with acoupled system of glucose dehydrogenase/nicotine adeninedinucleotide/diaphorase/tetrazolium salt and glucosedehydrogenase/dichlorophenolindophenol. A yellow component is introducedinto the colors obtained by the use of a background dye.

DE 32 11 167 claims at least two enzyme systems each of which isindependently capable of catalyzing the direct or indirect conversion ofa substrate. The specification defines "independent of one another" tomean that, in the simultaneous presence of the systems that react withthe substrate, the reaction through the second system takes place onlyafter the coenzyme of the first system has been largely consumed.

DE 32 47 894 discloses a test system and method for the determination ofreduced nicotine adenine dinucleotide, which produces an enlargedmeasuring range for the determination of NAD(P)H or substrates orenzymes reacting under the formation or consumption of NAD(P)H. Thesystem is characterized in that it contains simultaneously severalsubstances with different electrochemical potentials, functioningindependently of one another as electron acceptors for NAD(P)H.Particular examples of electron acceptors include 2,6-dichloroindophenoland INT, (2-(4-iodophenyl)-3-(4-nitrophenyl)-5 -phenyltetrazoliumchloride). The specification states that the test system can beimpregnated into absorbent materials. A yellow component is added to thecolor seen by incorporation of a background dye, titanium yellow.

Japanese Patent Application 59-106299 was published June 19, 1984. Theapplication discloses a method for estimating NAD(P)H with oxidizedglutathione in the presence of glutathione reductase and a color formingagent Examples of color forming agents given are 5,5'-dithiobis(2-nitrobenzoic acid), N-(1-anilino-naphthyl-4)maleimide,Beta-hydroxyethyl-2,4-dinitrophenyldisulfide, 2,2-dithiopyridine andbenzimidazolyl maleimide.

European Patent Application 0-153-872 discloses a method for thedetermination of the reduced form of nicotinamide adenine dinucleotide(phosphate) which involves reacting NAD(P)H with (1) peroxidase or thioloxide reductase and (2) diaphorase or an electron carrier in thepresence of a chromogen and determining the pigment thus formed. Thesetwo reactions do not act on a common substrate.

None of these disclosures provides a system composed of two independentcatalytic systems, one being a disulfide reductase system, which allowsthe production of a full spectrum RAINBOW, including a yellow componentgenerated in situ; the particular final hue produced depending on theconcentration of the analyte.

SUMMARY OF THE INVENTION

The invention provides a test composition for the visual determinationof the concentration of an analyte in a fluid sample, comprising: (a) acatalytic system capable of generating reduced nicotinamide adeninedinucleotide by reaction with the analyte of interest; (b) a firstindependent catalytic system capable of generating a reduced firstindicator by reaction with reduced nicotinamide adenine dinucleotide;and (c) a second independent catalytic system capable of generating achange of hue of a second indicator component by reaction with reducednicotinamide adenine dinucleotide, which second independent catalyticsystem includes

(i) a disulfide reductase;

(ii) a disulfide substrate; and

(iii) a thiol indicator, wherein the disulfide reductase is capable ofcatalyzing the reaction between reduced nicotinamide adeninedinucleotide and the disulfide substrate to produce a product which caninteract with the thiol indicator to produce the change of hue of thesecond indicator; whereby, the generation of the reduced first indicatorand the change of hue of the second indicator can be controlled toprovide a range of hues, the particular final hue produced by the testcomposition depending on the concentration of the analyte.

Preferably one system produces a yellow hue. In one preferred system,the thiol indicator is reduced to produce a yellow second indicator.

The composition can be dissolved to provide a test solution orincorporated in a carrier matrix to provide a test device format

A preferred embodiment is a glucose whole blood test device which iscapable of generating a full spectrum rainbow, including a yellow huegenerated in situ, the particular final hue of the test device dependingon the concentration of glucose in the test sample.

DESCRIPTION OF THE INVENTION

Visual color matching is convenient and it can provide an acceptablyaccurate determination of analyte concentration without the need forexpensive instrumentation. Color can be broken into components such assaturation, lightness and hue. Hue is commonly referred to as "color"e.g., whether something "looks" blue, red or yellow. Throughout thespecification, the term "hue" is used.

Generally, one hue is associated with one form of a single indicator.Therefore, in order to generate a range of hues, a number of differentindicator molecules are required. The possible changes of hue arenumerous An indicator can change from one hue to another, from one hueto colorless or from colorless to a hue. It is even possible that theindicator itself will not change hue but that the format of a deviceincorporating the test composition is such that there is an apparentchange in hue of the device when contacted with a test sample containingthe analyte. For example, the hue of the indicator could not be seenprior to the reaction of the test composition with the analyte but isvisible after that reaction occurs. Because it is desirable that thechange of final hue exhibited by the test device from one analyteconcentration to another be as clear to the user as possible, thechanges colorless to a hue and a hue to colorless have been preferred.When using two or more indicators, at least one indicator component ispreferably changed from one hue to colorless. Otherwise the final hue ofthe test composition or device will move toward black as more hues areproduced

It is particularly desirable to control the production and/ordisappearance of indicators which in one form exhibit one of the primaryreflective hues: red, yellow, blue. While a composition containing twoindicator components can be used, the use of three indicator componentshas been found to be advantageous.

For example, consider a test composition containing indicators whichexhibit the following change in hue in the order shown.

indicator 1) blue to colorless

indicator 2) colorless to yellow

indicator 3) colorless to red

If the indicators react in sequence, the apparent hue of the testcomposition would be blue to colorless to orange (yellow+red).

However, if indicator 2 changed hue concurrently with the change in hueof indicator 1, the change in hue of indicator 3 occurring later, theapparent hue of the test composition would be blue to green(blue+yellow) to yellow to orange (yellow+red) to red. A similar exampleis:

indicator 1) red to colorless

indicator 2) colorless to yellow

indicator 3) colorless to blue

If the indicators reacted in sequence, the apparent hue of the testcomposition would be red to colorless to yellow to green (blue+yellow).If the indicators reacted as described above, with two indicator changesbeing produced simultaneously, the third change being delayed, theapparent hue of the test composition would be red to orange (red+yellow)to yellow to green (yellow+blue) to blue.

Both of these schemes, containing simultaneous hue changes, couldproduce a full spectrum RAINBOW. Such a RAINBOW would be desirablebecause it would provide the widest range of color visible to the eyewhich could be used to make the differentiation between levels ofanalyte easier to interpret.

The problem is twofold: 1) to select the indicators having changes inhue induced independently to give the maximum change in hue, and 2) toensure that these indicators react in an orderly fashion depending onlyon the concentration of the analyte.

It has been found that a range of hues can be produced by using twoindependent catalytic systems, each reactive with reduced nicotinamideadenine dinucleotide (NADH) to produce one or more changes in hue. Thesystems react substantially simultaneously with NADH. The hue producedby a particular analyte concentration can be controlled by the relativeincrease or decrease of the concentrations of the components andcatalysts in the test composition.

Each independent catalytic system is a system capable of interactingwith one or more indicators in the presence of NADH to produce a changein hue of the indicator(s). One system contains at least an oxidizedindicator and a catalyst capable of facilitating the reduction of theoxidized indicator in the presence of NADH. The other system contains atleast a disulfide substrate, a thiol indicator and a disulfide reductase(the catalyst). The disulfide reductase system can function in threegeneral ways: 1) the thiol indicator can interact with the productformed by the reaction of NADH and the disulfide substrate to producethe change of hue of the second indicator; 2) the thiol indicator can bereduced to form a reduced second indicator; and 3) that secondindicator, in either case, can have a yellow hue. It has proven to beparticularly difficult to generate a yellow hue in situ.

The terms catalyst and catalytic are used in their conventional senseherein. A "catalytic" reaction is a reaction in which the rate ischanged by the addition of a "catalyst" but the catalyst itself isunchanged. The catalytic reactions referred to herein are usuallyenzymatic, but can also include nonenzymatic reactions such as thosecatalyzed with phenazine methosulfate. The term "independent" means thatthe oxidized indicator(s) being reduced in one catalytic system is notin equilibrium with the oxidized indicator(s) being reduced in the othercatalytic system during the reaction time involved

The NADH is generated from the analyte of interest by a catalytic systemwhich is usually enzymatic. Table 1 shows analytes of clinical interestand useful enzymatic systems which can produce NADH. These reactions andthe reaction components required are well known and are presently thebasis for many diagnostic reactions which generate changes in intensityof a single hue in response to analyte concentration. The enzyme systemcapable of generating NADH from the analyte of interest is commonly adehydrogenase system, although any system capable of generating NADH canbe used.

                  TABLE 1                                                         ______________________________________                                        analyte       enzyme                                                          ______________________________________                                        glucose       glucose dehydrogenase                                                         hexokinase/glucose-6-phosphate                                                dehydrogenase                                                   cholesterol   cholesterol dehydrogenase                                       alcohol       alcohol dehydrogenase                                           lactate       lactate dehydrogenase                                           ______________________________________                                    

The reduced form of nicotinamide adenine dinucleotide, NADH, has beenfound to be a particularly useful common substrate. Although thisspecification refers exclusively to nicotinamide adenine dinucleotide,NAD, and its reduced form NADH, it is to be understood that thedisclosure applies equally to the phosphorylated forms NAD(P) andNAD(P)H.

Catalytic systems useful for the common substrate NADH, based ondiaphorase or catalysts having diaphorase like activity, such asphenazine methosulphate and 1-methoxyphenazine methosulphate, will nowbe described in detail.

The diaphorase system has been found to be useful as one pathway becausetwo oxidized indicators are readily available which exhibit distinctchanges in hue. DCIP, 2,6-dichloroindophenol, is a blue compound whichis reduced to a colorless form in the presence of diaphorase and NADH.INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, iscolorless in the oxidized form but becomes red when reduced in thepresence of diaphorase and NADH. The two changes in hue aresubstanteously sequential. Other indicators reactive with diaphorase andNADH can be used. For instance,N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl}-2-chloro-6-sulfo-4-iminobenzoquinone, referred to for convenience asTR-1, and p-nitroblue tetrazolium chloride, referred to herein as NBT,can be substituted for DCIP and INT. Other indophenols and relatedsubstituted alkyl, nitro, halogen and pseudohalogen derivatives can besubstituted for DCIP, as well as other indicators of similar reductionpotential capable of being reduced in the presence of NADH. Othertetrazoliums can be used in place of INT. Many are known in the art andhave been catalogued in reviews such as " An Introduction to the Use ofTetrazolium Salts in Quantitative Enzyme Chemistry", F. P. Altman,Koch-Light Laboratories, Ltd., Colnbrook, England, 1972; and "TheChemistry of Formazans and Tetrazolium Salts", A. W. Nineham, Chem. Rev, 55:355 (1955).

A second parallel pathway is provided by the second independentcatalytic system based on a disulfide reductase. A number of reductasesystems are shown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        disulfide substrate                                                                           disulfide reductase                                           ______________________________________                                        L-cystine       cystine reductase                                             oxidized glutathione                                                                          glutathione reductase                                         lipoamide       dihydrolipoamide                                                              reductase (referred to                                                        as lipoamide dehydro-                                                         genase herein)                                                protein-disulfide                                                                             protein-disulfide                                                             reductase                                                     oxidized thioredoxin                                                                          thioredoxin reductase                                         CoAS-Sglutathione                                                                             CoAS-Sglutathione reductase                                   asparagusate    asparagusate reductase                                        ______________________________________                                    

The disulfide reductase system has three components: a disulfidesubstrate, a disulfide reductase capable of facilitating the reactionbetween the disulfide substrate and NADH, and a thiol indicator whichcan interact with the product of the reaction between the disulfidesubstrate and NADH to produce the change of hue of the second indicator.A preferred disulfide substrate is lipoamide and analogs thereof whichcan be used with lipoamide dehydrogenase. The disulfide reductase systemhas been found to be particularly useful for introducing yellow into therange of hues generated by the test composition.

The thiol indicator is any substance which will interact with a thiol(--SH compound) to give observable color Preferred thiol indicators arecolorless indicators which become yellow upon interaction. Commonly, thethiol indicator is an oxidized indicator which is reduced in thepresence of the product of the reaction between the disulfide substrateand NADH to produce a reduced second indicator which can be, but neednot be, yellow. However, other types of interaction which will producecolor are also contemplated. For example, thiol indicators can bechelating agents such as nitroprusside, which would interact with theproduct of the disulfide substrate/NADH reaction to produce a red hue. Apalladium complex can also be used to generate red A yellow hue can begenerated by interaction of the thiol indicator with the product of thedisulfide substrate/NADH reaction by alkylation if the product iscysteine Alternatively a cysteine product could be reacted withnoradrenachrome to produce a yellow hue.

Another general type of thiol indicator is a chromophore of the desiredhue, preferably yellow, immobilized behind an opaque barrier. Onreaction with the product of the disulfide substrate/NADH reaction, thechromophore is released from its attachment and is free to diffusethrough the opaque barrier to a position where it is visible to anobserver.

Commonly, the thiol indicators which undergo reduction are disulfidecompounds. Especially preferred are analogs of5,5'-dithiobis-(2-nitrobenzoic acid) referred to herein as DTNB, whosestructure is shown below: ##STR1## DTNB is commonly referred to asEllman's Reagent. Changes at the carboxylic acid hydroxyl group havebeen found to be useful. Analogs of DTNB, which are defined herein toinclude positional isomers of DTNB, of the structure shown below arepreferred: ##STR2## R can be many groups such as those providing anester or amide linkage, e.g., ##STR3##

While water solubilizing groups such as those shown above are preferredin gelatin matrix formats, water insoluble analogs can be used incompartmentalized formats, described later in the specification.

A preferred water soluble analog compound is3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl disulfide,structure shown below, whose synthesis is detailed in the examples.##STR4## The compound is referred to subsequently in the specificationas 3-ND for convenience. This compound is colorless in the oxidized formand becomes yellow in the presence of lipoamide, NADH and lipoamidedehydrogenase.

Hues of particularly useful indicators are shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                                     oxidized   reduced                                               ______________________________________                                        DCIP           blue         colorless                                         INT            colorless    red                                               TR-1           red          colorless                                         NBT            colorless    blue                                              DTNB           colorless    yellow                                            3-ND           colorless    yellow                                            EA1            colorless    yellow                                            ______________________________________                                    

It has been found that a color retardant can be added to thecomposition. The color retardant has essentially no affect on the huevisible to the user, but its inclusion can delay the catalytic affect ofthe independent systems, delaying the reduction of the oxidizedindicators and therefore changing the final hue produced for aparticular concentration of analyte. Useful color retardants includepotassium ferricyanide and 1-N-ethyl-4-methylquinolinum iodide andanalogs thereof or mixtures of these compounds, potassium ferricyanidebeing preferred. Color retardants effectively change the measurementrange of the composition.

Other components such as buffers, surfactants and polymers can be addedto the composition. The pH is generally chosen to give good performanceand stability to the reagents and is controlled by use of buffers. Theuse of buffers is preferred since the enzymes function better within thepH range of about 6 to 8. Choice of a buffer is within the skill of theart. Useful buffers include, but are not limited to,N-2-hydroxyethylpiperazine-N'-2 -ethanesulfonic acid (HEPES),2-[tris(hydroxymethyl)methyl]amino ethanesulfonic acid (TES),2-[N-morpholino]-ethanesulfonic acid (MES) and[3-(N-morpholino)propanesulfonic acid] (MOPS). Surfactants and polymerscan be particularly useful in formulations containing a cationictetrazolium salt as an oxidized indicator since surfactants such aspolyoxyethylene ether, available under the

trademark TRITON® X-100 from Sigma Chemical Co., and polymers such aspolyvinylalcohol and polyvinylpyrrolidone, available as PVP K30 fromAldrich Chemical Co., appear to help solubilize the cationic indicatorand prevent interaction with the other indicators in the testcomposition. Surfactants also improve wettability of the device in a dryphase formulation. Enzyme stabilizers, such as bovine serum albumin, canalso be added.

Test compositions of this invention can be used by dissolving thecomposition in a solution or they can be incorporated into a carriermatrix and affixed to a support member such as a polyester strip toprovide dry reagent strips which are well known in diagnostics. Thesestrips provide a format which is convenient to carry and store and whichis particularly useful to home users such a diabetics. Preferredcompositions of this invention generate a final hue, which can beassociated with a particular analyte concentration, in less than aboutten minutes.

The carrier matrix employed can be any of several known in the industry,as long as the matrix can be incorporated with the composition and itdoes not interfere with the reactions required for the production ofcolor. These include paper and films such as those made from naturalpolymers, latexes, polyurethanes, silicones or combinations of these.

In order to obtain the clearest colors possible, a clear carrier matrixwas preferred. Since common analytes for this invention are watersoluble compounds such as those found in body fluids, carrier matriceswhich can contain water, such as hydrophilic carriers, are preferred.Suitable hydrophilic carriers include agarose, gelatin,poly(vinyl)alcohol, poly(propylimine), carrageenan and alginic acid.Other carriers could be used. Mixed multilayer carriers composed of anabsorbent opaque matrix, such as paper, and a hydrophilic (e.g.,gelatin) carrier layer can be advantageously used when the testcomponents are compartmentalized.

In a preferred embodiment, a solution of 1.25% carbodiimide was used tocrosslink a multilayer gelatin carrier matrix. This provides a formatsuitable for a whole blood glucose test which allows the blood sample tobe removed from the test device by wiping the surface. Other coatingmaterials, well known in the art, can be used to allow the device to bewashed or wiped to remove a colored sample if necessary.

The hydrophilic carrier layer or layers are coated onto a rigid backingor support member such as polystyrene, polyester and the like. Thebacking can be opaque or transparent, although an opaque white backingis commonly preferred for visually read tests.

The number and types of components, which can be used in the independentcatalytic systems described previously, is increased by the use ofcompartmentalization of possibly incompatible components in a testdevice format. Compartmentalization can take on many forms. Componentscan be separated by placing some in a separate layer, by solubilizingwithin one phase of an emulsion, by precipitation, by encapsulation andso forth. Some of the available methods are described in detail in theExamples.

A multilayer gelatin carrier was preferred for compartmentalization. INTcould be placed in one layer away from the other indicator components.It was also found that the position of the components in various layerscould change the hue visible to the user at a particular analyteconcentration and therefore afforded another means of controlling thehue generated. The apparent hue of the device can be changed by changingthe order or thickness of layering.

A particular example showing the affect of compartmentalization ofcomponents in different layers on the hue visible to the user will nowbe described in detail.

A RAINBOW test device for the determination of glucose can be preparedwith the following independent catalytic reactions:

NADH generating system:

glucose (analyte)

glucose dehydrogenase (enzyme)

NAD (additional component)

First independent catalytic system

diaphorase (catalyst)

DCIP (oxidized first indicator, blue)

INT (oxidized third indicator, colorless)

DCIP was reduced by NADH first. Therefore the first independentcatalytic system produces a blue to colorless, then a colorless to red,change in the hue visible to the user.

Second independent catalytic system:

lipoamide (disulfide substrate)

lipoamide dehydrogenase (disulfide reductase)

DTNB (thiol indicator, oxidized second indicator, colorless)

Compartmentalization of the oxidized indicators, e.g., placing theindicators in different gelatin layers, can effectively change the hueseen at different glucose concentrations even though the concentrationsof test components are the same. As an example, three films were madewith the indicators arranged in different layers, but keeping theconcentrations of the components of the total test composition the same.##STR5##

In gelatin, certain water soluble molecules, such as DNTB, are capableof diffusing through the gelatin after contact with an aqueous sample,while others, such as INT, do not readily migrate.

In Film A, glucose reacts with the enzymes, then the NADH producedreacts with the independent catalytic systems to reduce DCIP and DTNB.However, reduction of INT is delayed until the NADH can diffuse into thelower layer of the film. At 250 mg/dL glucose, Film A is yellow orange.

In Film B, the glucose must diffuse through the INT layer beforereaching the enzymes to react and produce NADH. With the production ofNADH, the DCIP will be reduced but the reduction of INT is delayed sincethe NADH must diffuse back up onto the top layer before the INT can bereduced. Therefore, at 250 mg/dL glucose, Film B is yellow-green. Thecompartmentalization of INT above the enzymes and other indicatorcomponents has changed the color observed at this particularconcentration of glucose.

In Film C, the glucose reacts with the enzymes in the top layer. As theNADH migrates through the INT layer, a small amount of INT reducedcausing a slight red color. As the NADH migrates through to the bottomlayer, the DCIP and the DTNB are reduced. Finally, since not all theNADH has reacted due to the concentration of components chosen, the NADHwill react with the INT. At 250 mg/dL glucose, Film C is a reddishorange.

The ultimate goal of the invention was to generate distinctly differenthues at different analyte concentrations. It has been shown that thisgoal can be achieved with test compositions of this invention with avariety of methods as summarized below.

1) Choose oxidized indicators which will generate the desired hue orturn colorless upon reduction.

2) Choose indicators for reaction in a particular catalytic system whichhave differing reduction potentials which will control the sequence ofreactions in that system.

3) Control the quantities of components in the independent catalyticsystems so the components involved in one reaction sequence areessentially exhausted at chosen analyte concentrations.

4) Increase (or decrease) the amount of catalyst in a system. This willincrease (or decrease) the rate of interaction or reduction of theindicator components by that system and therefore change the apparenthue at a particular analyte concentration.

5) Compartmentalize the components of the reaction system in a testdevice. The advantages of compartmentalization can include the abilityto: change the apparent final hue of the device even when componentconcentrations are the same; utilize incompatible indicators or waterinsoluble indicators; and utilize enzyme systems normally inhibited bythiol indicators.

6) Add a color retardant which will delay the reactions of theindependent catalytic systems.

The methods suggested above can be combined.

The invention will now be illustrated, but is not intended to belimited, by the following examples:

EXAMPLES

    ______________________________________                                        Abbreviations                                                                 ______________________________________                                        MPMS          1-methoxyphenazine                                                            methosulphate                                                   PMS           phenazine methosulphate                                         DCIP          2,6-dichloroindophenol                                          TR-1          N-(2,3-dimethyl-5-oxo-1-                                                      phenyl-3-pyrazolin-4-                                                         yl)-2-chloro-6-sulfo-                                                         4-iminobenzoquinone                                                           (for preparation see                                                          Example 1A)                                                     INT           2-(4-iodophenyl)-3-(4-nitro-                                                  phenyl)-5-phenyltetra-                                                        zolium chloride                                                 NBT           nitroblue tetrazolium                                                         chloride                                                        DTNB          5,5-dithiobis(2-nitro-                                                        benzoic acid)                                                   3-ND          3-N-(3-dimethylamino                                                          propyl) carboxamido-                                                          4-nitro-phenyl di-                                                            sulfide (for prepara-                                                         tion see Example 1B)                                            EA1           Dibutyl-5,5'-dithiobis-                                                       (2-nitrobenzate)                                                              (for preparation see                                                          Example 2C)                                                     NAD.sup.+     Nicotinamide-adenine                                                          dinucleotide,                                                                 lithium salt                                                    HEPES         buffer, N-2-hydroxyethyl                                                      piperazine-N'-2-ethane                                                        sulfonic acid                                                   MES           buffer, 2-[N-morpholino)-                                                     ethanesulfonic acid                                             TAPSO         buffer, 3-(N-tris(hydroxy-                                                    methyl)methylamino)-                                                          2-hydroxypropane-                                                             sulfonic acid                                                   TRIS          buffer, Tris(hydroxyemthyl)-                                                  aminomethane                                                    Triton ® X-100                                                                          surfactant, polyoxyethyl-                                                     ene ether available                                                           from Sigma Chemical                                                           Co.                                                             GDH           glucose dehydrogenase                                                         (EC 1.1.147) capable                                                          of producing NADH                                               LipDH         lipoamide dehydrogenase                                         LDH           lactate dehydrogenase                                           U             International Units, a                                                        measure of enzyme                                                             activity (one U is                                                            the enzyme activity                                                           required to catalyze                                                          the conversion of one                                                         micromole of sub-                                                             strate per minute                                                             under specified condi-                                                        tions of temperature                                                          and pH                                                          PET           polyethylene terephthalate                                      FMN           flavin mononucleotide                                           PE 310        polyethylene coated paper                                       BSA           Bovine Serum Albumin                                            PVP K30       polyvinylpyrrolidone,                                                         molecular weight                                                              40,000, available                                                             from Aldrich                                                                  Chemical Co.                                                    dL            deciliters                                                      mL            milliliters                                                     μL         microliters                                                     g             grams                                                           mm Hg         millimeters of mercury,                                                       pressure designation                                            mp            melting point                                                   μ          microns                                                         RT            room temperature, usually 25° C.                         ______________________________________                                    

EXAMPLE 1 Preparation of Compounds A.N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-2-chloro-6sulfo-4-iminobenzoquinone(TR-1)

TR-1 is an indicator which is red in the oxidized form and is colorlessupon reduction. It has a reduction potential similar to DCIP and has abeen used to produce a "reverse" RAINBOW. TR-1 was prepared as follows:

Ammonium hydroxide (1 N, 10 mL) was added to a mixture of 0.4 g (1.9mmol) of 4-aminoantipyrine, and 0.5 g (1.7 mmol)2-hydroxy-3,5-dichlorobenzene sulfonic acid disodium salt in 50 mL ofwater. After stirring briefly, 1.3 g (3.3 mmol) of potassiumferricyanide (K₃ Fe(CN)₆) was added and the reaction allowed to stir atroom temperature for one hour. The mixture was filtered to yield 0.2 g(21%) of a dark brown solid. The product was homogeneous on thin layerchromatography (silica gel, with 4:1 chloroform/methanol) and apparentlywas a mixture of alkali and ammonium salts.

Analysis: Calculated for C₁₇ H₁₀ ClN₃ O₅ Na: C, 47.54; H, 3.04; N, 9.72.Found: C, 46.10; H, 3.61; N, 11.00.

¹ H NMR (D₆ DMSO) δ: 7.80-7.20 (m, 7H), 3.40 (s, 3H), 2.53 (s, 3H),

IR (KCl): 1660, 1630, 1400 cm⁻¹.

B. 3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl Disulfide (3-ND)

A water soluble analog of 5,5'-dithiobis-(2-nitrobenzoic acid) wasprepared for use as a thiol indicator. This is an indicator for use withthe disulfide reductase system which is colorless in the oxidized formand becomes yellow on reduction. The compound 3-ND, was prepared asfollows:

A suspension containing 7.93 g of 3-carboxy-4-nitrophenyl disulfide (20mmol), 1.2 mL of dry N,N-dimethylformamide (1.55 mmol), 11.7 mL ofthionyl chloride (60.3 mmol) and 400 mL of dichloromethane (CH₂ Cl₂) wasrefluxed for four hours and then was alllowed to stir overnight atambient temperature. The resulting clear solution was evaporated invacuo (12 mm followed by 0.1 mm Hg) to a yellow solid. The residue wasthen placed under an argon atmosphere, dissolved in 200 mL of CH₂ Cl₂,cooled to 0°, and then treated with 10.1 mL of3-dimethylaminopropylamine (80 mmol). The reaction mixture became a darkorange and a precipitate was formed. The resulting solution was allowedto warm to ambient temperature overnight. The reaction mixture was thensuccessively extracted four times with 200 mL portions of 5% sodiumbicarbonate solution, twice with 200 mL of water, and once with brine.The organic layer was then dried over magnesium sulfate, filtered, andconcentrated to give 9.39 g of a dark orange oil. The mixture was thenflash chromatographed on 500 g of SiO₂ -60 (70-230 mesh, available asSilica gel-60 from Merck & Co.) equilibrated and eluted with 50:10:1dichloromethane/methanol/concentrated ammonium hydroxide solventmixture. Fractions of 25 mL were collected. Fractions 110 to 205containing the purified product were pooled and concentrated in vacuo togive 7.06 g of an orange solid. The crude product was recrystallizedfrom ethyl acetate using a treatment with pulverized carbon black suchas norite and a diatomaceous earth, a filtering aid available fromManville Products Corp., Denver, Col., under the trademark Celite®.Obtained was 5.47 g of light yellow crystals after drying at 55°, 0.1mm. Yield=48.4%. mp 152°-156°.

Analysis: Calculated for C₂₄ H₃₂ N₆ O₆ S₂ : C, 51.05; H, 5.71; N, 14.88.Found: C, 51.05; H, 5.56; N, 14.62.

PMR (60MHz, CDCl₃) δ=1.73 (quintet, J=6Hz, 4H); 2.17 (s, 12H); 2.45 (t,J=6Hz, 4H); 3.53 (q, J=6Hz, 4H); 7.57 (s, 2H); 7.65 (dd, J=7Hz, 2Hz,2H); 8.02 (m, 2H, N--H); 8.03 (d, J=7Hz, 2H).

IR(KBr) cm⁻¹ : 3260, 3060, 2940, 2870, 2820, 2790, 650, 1560, 1530,1470, 1345.

Mass Spectrum (FAB) m/e: 565 (M+1, 13.6%).

C. Dibutyl-5,5'-Dithiobis-(2-nitrobenzoate) (EA-1)

This compound, which is referred to herein as EA-1, is a lipophilicanalog of 5,5'-dithiobis-(2-nitrobenzoic acid). It was prepared for useas a thiol indicator as follows:

A suspension containing 6.85 g (17.25 mmol) of5,5'-dithiobis-(2-nitrobenzoic acid), 3.46 mL of thionyl chloride, 0.346mL of N,N-dimethylformamide, and 350 mL of dichloromethane were heatedto reflux for 3 hours. Additional thionyl chloride (3.46 mL) andN,N'-dimethylformamide were added and refluxing was continued for 2hours. A clear, light green solution was obtained, indicating completeconversion to the bis-acid chloride. The solvents were removed in vacuoand the resulting light-green solid was placed under an Argonatmosphere. The residue was then suspended in 100 mL of pyridine andtreated with 20 mL of n-butanol. A mild exotherm and a darkened buthomogeneous reaction was obtained, which was allowed to stir overnight.The reaction solvents were removed in vacuo and the residue wasdissolved in 300 mL of chloroform. The organic layer was thensuccessively extracted thrice with 200 mL of 0.1 N HCl, twice with 200mL of 5% Na HCO₃ solution, and with 200 mL of brine. Drying (MgSO₄),filtration, and removal of solvent gave 12.79 g of an oil which wasdissolved in ethyl acetate and adsorbed in vacuo onto a small amount ofSiO₂ -60. The impregnated solid was then atop a column of 500 g of SiO₂-60 (230-400 mesh) which had been packed and equilibrated with 8:1hexane-ethyl acetate. The column was then flash chromatographed usingthis solvent mixture with fractions of 25 mL collected. Fractions190-270 were pooled and concentrated to give 8.07 g of product as an oilwhich solidified upon standing (92% yield).

Analysis: Calculated for: C, 51.96; H, 4.76; N, 5.08. Found: C, 52.49;H, 4.88; N, 5.45.

PMR (60 MHz, CDCl₃) δ: 0.97 (t, J=7Hz, 6H, CH₃ --CH₂); 1.2-1.9 (m, 8H,--CH₂ --CH₂ --); 4.37 (t, J=6Hz, 4H, --O--CH₂ --CH₂ --) 7.77 (s, 2H, C₆H); 7.83 (AB quartet, J=8Hz, 4H, C₃ H, C₄ H).

IR (KBr)cm⁻¹ : 1730 ##STR6##

Mass Spectrum: E.I. (70 EV) m/e =508 (37%, M⁺).

D. Dimethyl 5,5'-Dithiobis-(2-nitrobenzoate)

This compound is a lipophilic analog of 5,5'-dithiobis-(2-nitrobenzoicacid), which was prepared for use as a thiol indicator as follows:

A suspension containing 7.93 g (20 mmol) of5,5'-dithiobis-(2-nitrobenzoic acid), 1.2 mL of N,N-dimethylformamide,11.7 mL of thionyl chloride, and 400 mL of dichloromethane were refluxedfor 4 hours until a clear light green solution was obtained. Thesolvents were then evaporated in vacuo to obtain a yellow solid whichwas then placed under an Argon atmosphere, dissolved in 200 mL ofdichloromethane, and cooled to 0°. The stirred mixture was then treatedwith 4.03 mL of dry pyridine (50 mmol) and 30 mL of methanol. Theresulting mixture was then allowed to warm to ambient temperatureovernight. The reaction mixture was then successively extracted thricewith 300 mL of 5% NaHCO solution, thrice with 300 mL of 1 M citric acid,and 300 mL of brine. Drying (MgSO₄), filtration and removal of solventsgave 8.29 of a yellow solid which was recrystallized in two crops fromtoluene as a light yellow solid in 92% yield. mp 103°-104.5°.

Analysis: Calculated for: C, 45.28; H, 2.86; N, 6.60. Found: C, 45.74;H, 3.01; N, 6.25.

PMR (60 MHz, CDCl₃) δ: 3.93 (s, 6H, --O--CH₃); 7.8 (s, 2H, C₆ H); 7.83(AB quartet, J=8Hz, 4H, C₃ H, C₄ H).

IR (KRr) cm⁻¹ : 1740 ##STR7##

Mass Spectrum: EI (70EV) m/e: 424.3 (M⁺, 67.7%)

E. 3,6-Dioxaoctyl-5,5'-Dithio-(2-nitrobenzoate)

This compound is a water soluble analog of5,5'-dithiobis-(2-nitrobenzoic acid). It was prepared for use as a thiolindicator as follows:

A suspension containing 7.93 g (10 mmol) of 5,5'-dithio-(2-nitrobenzoicacid), 1.17 g of N,N-dimethylformamide, 11.7 mL of thionyl chloride, and400 mL of dichloromethane was heated to reflux with stirring for 2 hoursto obtain a clear solution. The solvents were removed in vacuo to give alight green solid which was placed under Argon, cooled to 0°, andsuspended with stirring in 120 mL of dry pyridine. Carbitol® (20 mL) wasthen added and the resulting mixture became an orangish-red homogeneoussolution after 1 hour reaction time (Carbitol® is a registered trademarkof Dow Chemical Co. and is chemically named 2-(2-ethoxyethoxy)ethanol).The mixture was then allowed to come to ambient temperature overnight.The sample was then evaporated in vacuo to a dark oil and flashchromatographed on 500 g of SiO₂ -60 (230-400 mesh) packed and elutedwith a 0.5% methanol-chloroform solvent mixture. Fractions of 25 mL werecollected. Fractions 150-186 were pooled and concentrated to give 7.86 gof a yellow oil. The sample was then dissolved in ether, treated with 4g of Norit, filtered through Celite, and precipitated as a dense yellowoil with hexane. The solvents were then decanted and the residualsolvents were removed in vacuo at 40° (0.1 mm) for 1 hour to give 5.48 gof a viscous, yellow oil (44% yield).

Analysis: Calculated for: C, 49.67; H, 5.13; N, 4.46. Found: C, 49.41;H, 5.09; N, 4.37.

PMR (60 MHz, CDCl₃) δ: 1.2 (t, J=7Hz, 6H, CH₃ --CH₂ --O--); 3.5 (q,J=7Hz, 4H, CH₃ --CH₂ --O--); 3.6 (s, 8H, --CH₂ --CH₂ --O--); 3.8##STR8## 7.8 (s, 2H, C₆ H); 7.82 (AB quartet, J=8Hz, 4H, C₃ H, C₄ H).

IR (CHCl₃)cm⁻¹ : 1740 ##STR9##

F. 3,6,9,12-Tetraoxadodecyl 5,5'-Dithio-(2-Nitrobenzoate) and1,12-Cyclic Diester

Both of these compound are water soluble analogs of5,5'-dithiobis-(2-nitrobenzoic acid). They were prepared for use asthiol indicators as follows:

A suspension containing 7.93 g (20 mmol) of5,5'-Dithiobis-(2-nitrobenzoic acid), 11.7 mL of thionyl chloride, and1.2 mL of N,N-dimethylformamide was heated to reflux for 2 hours toobtain a clear yellow solution. The solvents were removed in vacuo togive a light green solid, which was dissolved in a mixture of 50 mL ofdichloromethane and 5 mL of pyridine. This solution was then addedslowly to a 0° solution of 34.5 mL (38.8 g, 200 mmol) of tetraethyleneglycol in 100 mL of dichloromethane under an Argon atmosphere. Thereaction mixture was allowed to come to ambient temperature overnight.Evaporation of solvents in vacuo gave a brown oil which was partitionedbetween chloroform and water. The aqueous layer was extracted again withchloroform and the combined organic layers were washed with brine anddried Na₂ SO₄). Filtration and evaporation of solvent in vacuo gave anorange oil which was flash chromatographed on 500 g of SiO₂ -60 (230-400 mesh) packed and eluted with a 5% methanol-chloroform solventmixture. Fractions of 25 mL were collected. Fractions 24-38 were pooledand concentrated to give 2.21 g of a yellow glass. This was identifiedas the 1,12-cyclic-3,6,9,12-tetraoxaundecyl ester of5,5'-dithiobis-(2-nitrobenzoic acid). The yield of cyclic ester was 20%.

¹³ C NMR (22.5 MHz, CDCl₃) δ: 66.8, 68.3, 70.4 (all --O--CH₂ --CH₂--O--); 128.8, 142.4, 146.3 (aryl C); 164.3 ##STR10##

IR (CHCl₃)cm⁻¹ : 1730 cm⁻¹ ##STR11##

Fractions 91-101 were pooled and concentrated to give 3.94 g of theexpected 3,6,9,12-tetraoxadodecyl diester. (26% yield).

¹³ C NMR (22.5 MHz, CDCl₃) δ: 61.6, 65.6, 68.3, 70.2, 70.5, 72.4 (all--O--CH₂ --CH₂ --O--); 129.0, 142.4, 146.5 (aryl C); 164.5 ##STR12##

PMR (60 MHz, CDCl₃): integration ratio of aliphatic to aromaticprotons=5.6.

IR(CHCl₃)cm⁻¹ : 3600-3350 (--CH₂ --OH stretch): 1730 ##STR13##

EXAMPLE 2 Glucose Formulations

Glucose test devices useful for testing whole blood can be prepared asfollows. Reduced nicotinamide adenine dinucleotide was the commonsubstrate and was generated from glucose by glucose dehydrogenase. Thegeneral chemistry for RAINBOW glucose formulations is shownschematically on the following page. The thiol indicator, DTNB, iscommonly called Ellman's Reagent.

A. Pathway 1: Diaphorase/NBT/TR-1:

Pathway 2: LipDH/lipoamide/DTNB

    ______________________________________                                        Layer    Component          Quantity (g)                                      ______________________________________                                        1        Gelatin            1.13                                                       Water              6.78                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          0.40                                                       NBT                0.064                                             2        Gelatin            1.13                                                       HEPES Buffer, 1 M, pH 7.5                                                                        5.42                                                       Water              1.36                                                       PVP K30 (20%)      1.69                                                       K.sub.3 Fe(CN).sub.6                                                                             0.073                                                      DTNB               0.040                                                      GDH (64.6 U/mg)    0.060                                                      Diaphorase (118 U/mg)                                                                            0.005                                                      BSA                0.035                                                      Lipoamide          0.030                                                      LipDH (1350 U/mL)  200     μL                                              NAD.sup.+          0.060                                                      Mutarotase (5060 U/mL)                                                                           100     μL                                              Triton ® X-100 (4%)                                                                          0.4                                               3        Gelatin            1.13                                                       Water              6.78                                                       Triton ® X-100 (4%)                                                                          0.40                                                       TR-1               0.060                                             4        carbodiimide       1.25%                                             ______________________________________                                         ##STR14##

Procedure: The components of each solution were combined at 40° in theorder given. The solutions were degassed before coating. Layer 1 wasspread onto a polyester backing and dried. Layer 2 was spread onto Layer1 and dried and so on. The final device was made up of three gelatinlayers on a polyester backing. The carbodiimide coating provided ahardened surface by crosslinking the gelatin, which allows wiping thesurface of the device.

Dose Response: With increasing glucose concentration from 50 to 600mg/dL, the color sequence was pale red to olive green to blue black.

B. Pathway 1: MPMS/DCIP/INT

Pathway 2: Glutathion Reductase/Glutathione/DTNB

    ______________________________________                                        Layer    Component          Quantity (g)                                      ______________________________________                                        1        Gelatin            1.13                                                       Water              6.78                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          0.40                                                       INT                0.064                                             2        Gelatin            1.13                                                       HEPES Buffer, 1 M, pH 7.5                                                                        5.42                                                       Water              1.36                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          0.40                                                       DCIP               0.0147                                                     K.sub.3 Fe(CN).sub.6                                                                             0.073                                                      DTNB               0.040                                                      GDH (64.6 U/mg)    0.060                                                      NAD.sup.+          0.060                                                      MPMS               0.010                                                      Glutathione        230     μL                                              Reductase (2320 U/mL)                                                         Glutathione        0.089                                                      Mutarotase (5060 U/mL)                                                                           100     μL                                     3        Carbodiimide       1.25%                                             ______________________________________                                    

The procedure used for the formulation and testing of the device wasthat of Example 2A.

Dose Response:

    ______________________________________                                        Glucose (mg/dL)    Color                                                      ______________________________________                                        110                Peacock Blue                                               140                Green-Blue                                                 180                Dull Light Aqua                                            250                Dull Rose                                                  300                Dull Rose                                                  400                Rose                                                       500                Rose                                                       600                Dark Rose                                                  800                Burgandy                                                   ______________________________________                                    

The hues, blue to rose to burgundy, can be easily distinguishedvisually.

C. Pathway 1: MPMS/DPIP/INT

Pathway 2: LipDH/lipoamide/DTNB

    ______________________________________                                        Layer    Component          Quantity (g)                                      ______________________________________                                        1        Gelatin            1.13                                                       Water              6.78                                                       PVP K30 (20%)      1.69                                                       Triton X-100 (4%)  0.40                                                       INT                0.064                                             2        Gelatin            1.13                                                       HEPES Buffer, 1 M, pH 7.5                                                                        5.92                                                       Water              1.86                                                       PVP K30 (20%)      1.69                                                       Triton X-100 (4%)  0.40                                                       K.sub.3 Fe(CN).sub.6                                                                             0.073                                                      DTNB               0.040                                                      GDH (64.6 U/mg)    0.060                                                      NAD.sup.+          0.060                                                      MPMS               0.010                                                      DCIP               0.0147                                                     Lipoamide          0.030                                                      Mutarotase (5060 U/mL)                                                                           100     μL                                              LipDH (1350 U/mL)  200     μL                                     3        Carbodiimide       1.25%                                             ______________________________________                                    

The procedure used for the formulation and testing of the device wasthat of Example 2A

Dose Response:

    ______________________________________                                        Glucose (mg/dL)      Color                                                    ______________________________________                                         20                  Peacock Blue                                              40                  Peacock Blue                                              70                  Aqua                                                     110                  Teal Green                                               140                  Mint Green                                               180                  Sea Green                                                250                  Green Gold                                               300                  Gold                                                     400                  Orange Gold                                              500                  Orange                                                   600                  Dark Orange                                              800                  Darker Orange                                            ______________________________________                                    

The colors blue, green, gold, orange are visually distinct.

D. Pathway 1: DCIP/INT/MPMS

Pathway 2: LipDH/lipoamide/3-ND

    ______________________________________                                        Layer    Component          Quantity (g)                                      ______________________________________                                        1        Gelatin            1.13                                                       Water              6.78                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          0.40                                                       INT                0.064                                             2        Gelatin            1.13                                                       HEPES Buffer, 1 M, pH 7.5                                                                        5.92                                                       Water              1.86                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          0.40                                                       K.sub.3 Fe(CN).sub.6                                                                             0.073                                                      DCIP               0.0147                                                     3-ND (20 mM)       1.25    mL                                                 GDH                0.060                                                      NAD.sup.+          0.060                                                      MPMS               0.010                                                      Lipoamide          0.030                                                      Mutarotase (5060 U/mL)                                                                           100     μL                                              LipDH (1350 U/mL)  200     μL                                     3        Carbodiimide       1.25%                                             ______________________________________                                    

The procedure used for the formulation and testing of the device wasthat of Example 2A.

Dose Response:

    ______________________________________                                        Glucose (mg/dL)      Color                                                    ______________________________________                                         40                  Peacock Blue                                              70                  Teal Green                                               110                  Mint Green                                               140                  Light Mint                                               180                  Sea Green                                                250                  Tan                                                      300                  Dark Tan                                                 400                  Pale Brown                                               500                  Rust                                                     600                  Rust                                                     800                  Dark Red                                                 ______________________________________                                    

With this formulation, using the DTNB derivative 3-ND, the generation ofthe final hue of the test device, visible to the user, was complete in 8minutes.

E. Pathway 1: MPMS/DCIP/INT

Pathway 2: LipDH/lipoamide/3-ND

    ______________________________________                                        Layer    Component           Quantity (g)                                     ______________________________________                                        1        gelatin             1.13                                                      water               6.78                                                      PVP K30 (20%)       1.69                                                      Triton ® X-100 (4%)                                                                           .40                                                       INT                 .064                                             2        gelatin             1.13                                                      HEPES Buffer, 1 M pH = 7.5                                                                        5.92                                                      water               1.86                                                      PVP K30 (20%)       1.69                                                      Triton X-100 (4%)   .40                                                       3-ND (40 mM)        2.50    mL                                                DCIP                0.0147                                                    GDH                 0.060                                                     NAD.sup.+           0.060                                                     MPMS                0.010                                                     lipoamide           0.030                                                     mutarotase (5060 U/mL)                                                                            100     μL                                             LipDH (1350 U/mL)   200     μL                                             K.sub.3 Fe(CN).sub.6                                                                              0.055                                            3        carbodiimide        1.25%                                            ______________________________________                                    

Dose Response:

    ______________________________________                                        Glucose mg/dL        Hue generated                                            ______________________________________                                         0                   blue                                                      20                  dark blue green                                           40                  blue green                                                70                  light green                                              110                  yellow green                                             140                  gold                                                     180                  gold orange                                              250                  orange                                                   400                  red orange                                               800                  deep red                                                 ______________________________________                                    

This device exhibits a full spectrum RAINBOW; blue, green, gold, orange,red. The hue visible to the user depends on the concentration of glucoseand is generated in 7 to 8 minutes.

F. Whole blood glucose RAINBOW test device.

Pathway 1: Diaphorase/DCIP/INT

Pathway 2: LipDH/lipoamide/3-ND

    ______________________________________                                        Layer     Component          Quantity (g)                                     ______________________________________                                        1         Gelatin            1.13                                                       Water              6.78                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          .40                                                        INT                .064                                             2         Gelatin            1.13                                                       MES buffer, 1 M (pH-6.5)                                                                         5.92                                                       Water              1.86                                                       PVP K30 (20%)      1.69                                                       Triton ® X-100 (4%)                                                                          .40                                                        3-ND (40 mM)       2.5     mL                                                 GDH (64 U/mg)      .060                                                       NAD+               .060                                                       Diaphorase (118 U/mg)                                                                            .010                                                       BSA                .040                                                       Mutarotase (5060 U/mL)                                                                           100     μL                                              LipDH (1350 U/mL)  200     μL                                              DCIP               0.0147                                                     K.sub.3 Fe(CN).sub.6                                                                             0.055                                            3         Carbodiimide       1.25%                                            ______________________________________                                    

The procedure used for formulation of the device was that described inExample 2A. Testing was done with whole blood samples spiked to thedesired glucose levels, analyzed with a YSI glucose analyzer. The doseresponse data is given below. The test device, containing a testcomposition of the invention compartmentalized by layering in a gelatincarrier, exhibited a full spectrum RAINBOW over a glucose concentrationof 0 to 800 mg/dL.

The composition was designed to produce a green hue covering the normalblood glucose range.

Dose Response:

    ______________________________________                                        Glucose mg/dL        Hue Generated                                            ______________________________________                                         0                   blue                                                      40                  light blue                                                70                  blue green                                               110                  sea green                                                140                  pale green                                               180                  light yellow                                             250                  yellow orange                                            400                  orange red                                               800                  red                                                      ______________________________________                                    

G. Rainbow: Varying enzyme concentration.

The following example was designed to show how the final hue producedcan be controlled by changing the concentrations of the enzymes.

Pathway 1: Diaphorase/DCIP/INT

Pathway 2: LipDH/lipoamide/3-ND

    __________________________________________________________________________                     Quantity (g)                                                 Layer                                                                             Components   A      B      C      D                                       __________________________________________________________________________    1   Gelatin      1.13   1.13   1.13   1.13                                        Water        6.78   6.78   6.78   6.78                                        PVP K30 (20%)                                                                              1.69   1.69   1.69   1.69                                        Triton ® X-100 (4%)                                                                    .40    .40    .40    .40                                         INT          .064   .064   .064   .064                                    2   Gelatin      1.13   1.13   1.13   1.13                                        Hepes buffer, 1 M pH 7.5                                                                   5.92   5.92   5.92   5.92                                        Water        1.86   1.86   1.86   1.86                                        PVP K30 (20%)                                                                              1.69   1.69   1.69   1.69                                        Triton ® X-100 (4%)                                                                    .40    .40    .40    .40                                         3-ND (40 mM) 2.5 mL 2.5 mL 2.5 mL 2.5 mL                                      DCIP         0.0147 0.0147 0.0147 0.0147                                      GDH          .060   .060   .060   .060                                        NAD+         .060   .060   .060   .060                                        Diaphorase (118 U/mg)                                                                      .010   .010   .010   .025                                        Lipoamide    .030   .030   .030   .030                                        BSA          .040   .040   .040   .040                                        Mutarotase (5060 U/mL)                                                                     100 μL                                                                            100 μL                                                                            100 μL                                                                            100 μL                                   LipDH (1350 U/ml)                                                                          50  μL                                                                            200 μL                                                                            800 μL                                                                            200 μL                               3   Carbodiimide 1.25%  1.25%  1.25%  1.25%                                   __________________________________________________________________________

The films were prepared and tested as described in example 2A. Theresults are shown in the following table.

    __________________________________________________________________________    Glucose                                                                       mg/dL                                                                                A       B       C       D                                              __________________________________________________________________________     0   medium blue                                                                           medium blue                                                                           medium blue                                                                           dark blue                                         20  medium blue                                                                           light blue                                                                            medium blue                                                                           dark blue                                         40  medium blue                                                                           light blue                                                                            light blue                                                                            dark blue                                         70  light blue                                                                            aqua    aqua    medium blue                                      110  aqua    light green                                                                           light green                                                                           aqua                                             140  light aqua                                                                            light green                                                                           yellow beige                                                                          light aqua                                       180  peach beige                                                                           light yellow-                                                                         yellow gold                                                                           greyish                                                       peach           green blue                                       250  light rust                                                                            dirty gold                                                                            orange gold                                                                           dark peach                                       400  rust orange                                                                           rust orange                                                                           orange  burnt orange                                     800  dark rust                                                                             dark red                                                                              dark rust                                                                             deep red                                              orange  rust    orange                                                   __________________________________________________________________________

EXAMPLE 3 Paper RAINBOW

Although multilayered gelatin is a preferred matrix for a rainbowdevice, a device can be prepared using paper as a carrier. A solution ofthe following composition, made in the order shown was prepared.

    __________________________________________________________________________                   Triton                                                         Reagent    PVA X-100                                                                              Buffer                                                                             INT  DCIP                                                                              DTNB                                        __________________________________________________________________________    Concentration                                                                            10  10   1000 5    5   10                                          (mM) Stock Solution                                                           Volume (μL)                                                                           750 100  1000 1500 900 1500                                        Used                                                                          Final Concen-                                                                            1.24                                                                              0.17%                                                                              166  1.24 0.74                                                                              0.68                                        tration (mM)                                                                  Order of Mixing                                                                          1   2    3    4    5   6                                           __________________________________________________________________________            Reagent    MPMS  Gluthatione                                                                         Glut-Reductase                                 __________________________________________________________________________            Concentration                                                                            0.05  5     1790                                                                              U/mL                                               (mM) Stock Solution                                                           Volume (μL)                                                                           30    150   112                                                    Used                                                                          Final Concen-                                                                            250                                                                              nM 124                                                                              μM                                                                            33  U/μL                                            tration                                                                       Order of Mixing                                                                          7     8     9                                              __________________________________________________________________________

The buffer was TAPSO, pH 7.4. Both polyvinylalcohol (PVA) and the orderof reagent addition were critical in this example. A concentration of 1to 1.5% PVA prevents the coprecipitation of DCIP and INT in a papermatrix.

Whatman 31 ET paper was impregnated with this solution and dried at 50°C. The dried paper was cut and mounted on a plastic backing to form teststrips. The test strips were assayed with NADH solution and the resultsshown below.

    ______________________________________                                        NADH Concentration (mM)                                                       Time                                                                          (minutes)                                                                            0      .33      1       3.33  10                                       ______________________________________                                        1      blue   light blue                                                                             blue/green                                                                            brown brown/orange                             2      blue   light blue                                                                             pale green                                                                            brown orange                                   3      blue   light blue                                                                             faint green                                                                           brown orange                                   ______________________________________                                    

EXAMPLE 4 NADH Generating Systems Containing Enzymes Sensitive to ThiolReagents

A general problem with the described thiol detection reagent system isthat thiol indicators can react with protein systems, frequently leadingto inactivation of enzyme. Many such enzyme inhibitions by DTNB aredescribed in the literature. There are two general solutions, bothinvolving compartmentalizing the thiol reagent: (1) sequester the thiolindicator in an organic phase or as insoluble particles; (2) physicallyseparate the sensitive enzyme and the thiol reagent by immobilizing thelatter.

A. Approach (1)--Sequestering the Reagent.

A water insoluble analog of DTNB, EA1 was used (for preparation seeexample 1C).

There are two approaches to sequestering the reagent: (a) EA1 wasdissolved in an oil and dispersed as an emulsion in a gelatin film.

Organic Phase: EA1 (300 mM) was dissolved in tricresyl phosphate withtrioctylamine (1%).

    ______________________________________                                        Aqueous Phase       Final Concentration                                       ______________________________________                                        Gelatin             10%                                                       Potassium Phosphate,                                                                              50       mM                                               pH 7.5                                                                        DCIP                1.5      mM                                               ______________________________________                                    

The oil phase was emulsified with the aqueous phase in a Waring Blenderto a final concentration of 5%. Lipoamide dehydrogenase (Sigma type III,13.3 U/mL) was added to the emulsion. The emulsion was coated on aplastic support (170μ wet thickness) and dried at room temperature.

Treatment of the dried film with NADH/Lipoamide (approximately 1 mM) atpH 8.5 (tris buffer) produced a very rapid bleaching of the blue hue ofDCIP and formation of yellow by the EA1 reagent. Without NADH there wasno reaction.

The enzyme lactate dehydrogenase (LDH), which LDH, approximately 150U/mL, and lipoamide (approximately 1 mM) in about 250 mM lactate, pH8.5, produced a yellow hue in about 10 minutes. The blank, without LDH,was uncolored. The conclusion is that the use of an oil soluble thiolindicator, if compartmentalized in an oil emulsion, permits the use ofenzymes which are commonly believed to be sensitive to thiol reagents.

(b)--EA1 deposited directly into paper.

EA1 was dissolved in toluene to a concentration of 5.7 mM. Whatman 31 ETpaper was impregnated with this solution and dried at 50°. A secondsolution was prepared of the following constituents:

    ______________________________________                                                            Final                                                                         Concentration                                             ______________________________________                                        Triton ® X-100    0.1%                                                    (Tris).sub.2 sulphate pH 8.5                                                                        0.2     M                                               PVA (98.5% hydrolysed)                                                                              0.62%                                                   MPMS                  250     mM                                              Lipoamide             0.25    mM                                              LipDH                 30      U/mL                                            NAD                   0.5     mM                                              DCIP                  0.8     mM                                              ______________________________________                                    

(Tris)₂ sulphate is Tris buffer adjusted to the desired pH with sulfuricacid.

The dried paper was dipped into the second solution and dried again at50° C. and affixed to a plastic support.

Test solutions for LDH contained L-lactate (167 mM), (Tris)₂ sulphate,pH 8.5, (0.33 M) and LDH enzyme (rabbit muscle, Sigma type II) as shownin the following table with the test results.

    ______________________________________                                        LDH Concentration (μ/mL)                                                   Time                                                                          (minutes)                                                                            0        0.333    0.666  1.667   3.333                                 ______________________________________                                        3      dark blue                                                                              mid-blue pale blue                                                                            blue/green                                                                            mustard                               4      dark blue                                                                              mid-blue very pale                                                                            bluish  yellow                                                         blue   yellow                                        ______________________________________                                    

Very good color discrimination is seen between the various levels ofLDH.

Test Solution for alcohol detection

The ability of this compartmentalized film format to handle assaysrequiring the use of an enzyme which is sensitive to thiol reagents wasfurther tested using alcohol dehydrogenase.

Test solutions were prepared which contained various concentrations ofethanol, (Tris)₂ sulphate buffer, 0.5 M, pH 8.5, and alcoholdehydrogenase (from Baker's yeast, Sigma Catalog #A7011, 22.5 U/mL).

The test strips prepared as described above were used. The results areshown in the following table.

    __________________________________________________________________________    Ethanol Concentration (mg/dL)                                                 Time                                                                          (minutes)                                                                          0    1.84 3.22   4.6  6.9   11.5                                         __________________________________________________________________________    3    dark blue                                                                          less blue                                                                          medium blue                                                                          pale blue                                                                          blue/green                                                                          mustard                                      4    dark blue                                                                          less blue                                                                          medium blue                                                                          pale blue                                                                          mustard                                                                             yellow                                       __________________________________________________________________________

Very good discrimination was seen between levels of alcohol.

From these experiments it was concluded that the RAINBOW systemutilizing a thiol detection reagent can be used with thiol reagentsensitive enzymes.

B. Approach 2: Physical separation of thiol reagent sensitive enzyme andthiol reagent by immobilizing the latter.

DTNB can be covalently bound to a large molecule or can be physicallytrapped in a matrix, such as gelatin, which only allows penetration ofsmall molecules. Therefore direct interaction between the enzyme andthiol reagent would not be expected.

Preparation of immobilized DTNB

DTNB was linked to human serum albumin through the carboxyl functionusing a water soluble carbodiimide reagent.

A 30% human serum albumin solution was made 0.2 M in sodium phosphateand the pH adjusted to 4.57. It was then diluted to 4.8% albumin. DTNB(10.7 mM) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC, 10.7mM) were dissolved in a small amount of ethanol and added, with vigorousstirring, to the albumin solution maintained at 0° C. After 2 to 3hours, the lightly gelled material was transferred to dialysis tubingand dialysed extensively against water. Then it was lyophilized.

The crusty yellow solid was pulverized to a fine powder and dispersed in10% gelatin to a concentration of 1%. A coating was made (170μ), anddried at room temperature.

The film was evaluated with the following test mixture:

    ______________________________________                                                           Final                                                                         Concentration                                              ______________________________________                                        lithium lactate      250     mM                                               potassium phosphate  250     mM                                               buffer, pH 7.5                                                                glutathione          0.5     mM                                               glutathione reductase                                                                              9       U/mL                                             NAD                  0.2     mM                                               LDH                  5       U/mL                                             ______________________________________                                    

When contacted with this solution, the film produced a clear yellowcolor within one minute. A control solution, without LDH, produced nocolor.

The conclusion is that immobilizing the thiol reagent allows assay withthiol reagent sensitive enzymes.

The overall conclusion is that although many enzymes, such as alcoholdehydrogenase and cholesterol dehydrogenase are inhibited by DTNBaccording to the literature, there are many ways of avoiding thisinhibition. Consequently, the second catalytic system which involvesthiol indicators is generally applicable to assay of NADH as anintermediate and could be used to determine alcohol or cholesterol asanalytes.

EXAMPLE 5 Diffusible/Nondiffusible Dye Reaction

Another approach to generating a RAINBOW is where the hue of the testcomposition is made to appear as a result of diffusion. For instance, ifan indicator is unable to diffuse due to being covalently linked to amatrix, and this matrix is covered with an opaque layer, then theindicator will be invisible from the top. If, as a result of a reaction,the covalent linkage of the indicator to the matrix is severed, then theindicator can diffuse up through the opaque covering and become visible.

As an example, a film was prepared as described in Example 4B, whereDTNB was covalently bound to albumin which was then incorporated into agelatin matrix. Whatman 31 ET was impregnated with a solutioncontaining:

    ______________________________________                                                           Final                                                                         Concentration                                              ______________________________________                                        potassium phosphate, 160     mM                                               pH 7.5                                                                        glutathione          0.4     mM                                               gluthathione reductase                                                                             36      U/ML                                             ______________________________________                                    

The impregnated paper was dried at 50° C. Pieces of the dried paper werecut and laid on the gelatin film. The paper was quite opaque. An NADHsolution was added to the multilayer device prepared in this way andwater to another device to be used as a control. Within approximately 20seconds, the device contacted with NADH began to turn yellow and rapidlydeveloped to a deep yellow. The control pad had no color.

An LDH assay was made with the following solution:

    ______________________________________                                                           Final                                                                         Concentration                                              ______________________________________                                        potassium phosphate, 250     mM                                               pH 7.5                                                                        lithium lactate      250     mM                                               NAD                  0.2     mM                                               ______________________________________                                    

LDH (about 5 U/mL) was added to this solution and 30 μL of the solutionwas added to one of the multilayer paper/gelatin device described above.

After about 51/2 minutes, the LDH pad showed a faint but definite yellowcolor. The control, without LDH, had no color.

The conclusion is that indicators can indeed be made diffusible as afunction of analyte concentration.

In this approach, the indicator molecule is a combination of a huedetermining (chromophoric) part and reactive (cleavable) part. These canbe electronically isolated such that cleavage of the anchoring linkagedoes not significantly change the hue of the indicator. The amount, orintensity, of the indicator can be controlled by the activity of thecatalytic system involved in the reductive cleavage and the hue which isgenerated is determined independently by the chromophoric part of themolecule. This is a considerable advantage as it can be difficult tofind indicators with a suitable combination of hue and reactivity togenerate the desired final hue.

Other matrices to which a thiol indicator could be immobilized byoxidation are: thiolagarose or any protein using a bifunctional reagente.g., Lomant's reagent, dithiobis(succinimidyl propionate).

EXAMPLE 6

A test solution having the following composition was prepared. Reagentswere added in the order given, and it was important to have a polymer(polyvinylalcohol) present to avoid coprecipitation of INT and DCIP.

    __________________________________________________________________________               Triton                          Glutathione                        Reagent PVA                                                                              X-100                                                                             Buffer                                                                            INT DCIP                                                                              DTNB                                                                              MPMS  Glutathione                                                                         Reductase                                                                            Water                       __________________________________________________________________________    Order    1  2    3 4   5   6   7     8     9                                  Stock Solution                                                                         10%                                                                              10%                                                                              1000                                                                              5   5   10  .05   5     2270                                                                             u/mL                            concentration                                                                 (mM)                                                                          Volume added                                                                          375                                                                              50   500                                                                              750 450 750 25    75    57     3.03 mL                     (μl)                                                                       Final concen-                                                                         .62%                                                                             .08%                                                                               83 0.62                                                                              .37 1.24                                                                              .206                                                                             μM                                                                            .062  21 U/mL                            tration (mM)                                                                  __________________________________________________________________________

The buffer was Tris-Cl, pH 7.1. Glutathione reductase was from Sigma,catalog number G4751.

The test solution was reacted with NADH at the concentrations shown.Colors were estimated at 5 minutes after NADH addition. A controlsolution was identical to that above but without the glutathionereductase.

    ______________________________________                                        NADH                                                                          Concen-                                                                       tration                                                                       (mM)   0      .392     .95       1.82  3.33 6.4                               ______________________________________                                        Control                                                                              blue   light blue                                                                             blue/grey mauve red                                    Test   blue   green    emerald green                                                                           brown red  red                               Reagent                                                                       ______________________________________                                    

Many variations and modifications can be made from these exampleswithout departing from the scope or spirit of the invention.

What is claimed is: 1.3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl disulfide.